Our project is aimed at understanding the mechanism of protein degradation by studying mammalian cells with mutations in this function. These cells will be obtained in two ways: a. Selection of cultured, mutagenized normal cells for those whose inability to degrade protein prevents them from providing endogenous amino acids for protein synthesis. Cells of this sort should synthesize protein at a slower than normal rate and be protected against the lethal results of incorporation of radiolabeled amino acids and toxic amino acids analogues. b. Screening of cultured cells derived from patients with hereditary metabolic diseases suspected of affecting protein degradation either directly or indirectly. Having succeeded in obtaining mutants, we will characterize them genetically and biochemically to identify the functions needed for normal protein degradation which they lack. Our progress thus far is in two areas: a. We have worked out culture conditions allowing efficient induction of mutation, incorporation of 3H-amino acids under defined conditions, prediction of the toxicity of that treatment to wild type cells, and efficient screening of survivors of multiple treatment for their rates of protein synthesis and degradation. These procedures are now being applied in large-scale experiments. b. We have studied various aspects of protein degradation and its regulation in our parental cell type, cultured CHO cells. From these studies we have obtained evidence that the stimulation of degradation caused by amino acid starvation reflects regulation at the level of aminoacylation of tRNA.